Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A, B RT-qPCR showing that IMR1 and CB103 inhibits NOTCH1 and HES1 gene expression in LPS246 cells (n = 3). C, D Representative dose-response curves of LPS246 to varying concentrations of IMR1 and CB103, and the corresponding and IC50 values (n = 6). E, F Relative levels of Notch related genes in mLPS1 cells treated with IMR1 or CB103 (n = 3). G, H Representative dose-response curves of mLPS1 to varying concentrations of IMR1 and CB103 and the corresponding IC50 values (n = 6). I Clone formation assay of mLPS1 cells treated with vehicle control (DMSO), IMR1, or CB103 (n = 3) (left), and the quantification of colony area (right). J, K Effect of IMR1 and CB103 on mRNA levels of cancer stem cell markers in mLPS1 cells (n = 3). Data are presented as mean ± SD, *P < 0.05, **P < 0.001.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Quantitative RT-PCR, Gene Expression, Tube Formation Assay, Control

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A Cell proliferation of TetO-Empty vector and TetO-mPGC1αOE stable transfected mLPS1 cells treated with doxycycline (dox) or vehicle control (veh) (n = 3). B RT-qPCR analysis of the relative mRNA levels of cancer stem cell markers in Veh or Dox treated TetO-mPGC1αOE mLPS1 cells (n = 3). C The mLPS1 TetO-mPGC1αOE cells and mLPS1 TetO-Empty vector cells were subcutaneously transplanted into NRG mice and treated with Dox via drinking water (n = 5) for 21 days. D Tumor growth curves based on tumor volume calculation obtained from caliper measurement. E Morphology and (F) average weight of the transplanted tumors. G RT-qPCR analysis of PGC-1α expression in the transplanted tumors. H Immunofluorescent staining shows the relative expression of PGC-1α and Ki67 in the mLPS1 TetO-Empty vector and the mLPS1 TetO-mPGC1αOE allograft tumors. Nuclei were stained by DAPI. I Quantitation of Ki67 positive cells in H. Data are represented as mean ± SD. *P < 0.05, **P < 0.001. J Graphic illustration of how Notch signaling regulates cancer cell differentiation and mitochondrial function in liposarcoma.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Plasmid Preparation, Transfection, Control, Quantitative RT-PCR, Expressing, Staining, Quantitation Assay, Cell Differentiation

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A CRISPR targeting strategy to ablate the Rosa-NICDOE cassette in mLPS1 cells. The NICD cDNA containing exon 28 to 34 of Notch1 gene was inserted into the Rosa26 locus. Two guide RNAs (gRNAs) each spanning two exons were designed to target the NICDOE transgene without affecting the endogenous Notch1 gene. The sequencing results validated the correct targeting. B Relative mRNA levels of Notch related genes in mLPS1 and mLPS1 NICD knockout (mLPS1ΔNICD) cells (n = 3). C NICD and GFP protein levels in mLPS1 and mLPS1ΔNICD cells. D Cell proliferation of mLPS1 and mLPS1ΔNICD cells (n = 5). E Colony formation assay of mLPS1 and mLPS1ΔNICD cells (n = 3). Representative pictures of colony size (left) and quantification of colony area (right) were shown. F RT-qPCR analysis of mesenchymal stem cell markers Cd73, Cd90, and Cd105 in mLPS1 and mLPS1ΔNICD cells (n = 3). G Tumorigenicity of mLPS1 and mLPS1ΔNICD cells after subcutaneous transplantation into the left and right flanks of NRG mice, respectively (n = 5). H Images of the grafted tumors after surgical removal from the NRG recipient mice. I The average weights of the tumors (n = 5). Data are presented as mean ± SD, *P < 0.05, **P < 0.001.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: CRISPR, Sequencing, Knock-Out, Colony Assay, Quantitative RT-PCR, Transplantation Assay

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A RT-qPCR analysis showing expression of mature adipocyte marker genes in mLPS1 and mLPS1ΔNICD cells. B Fluorescent staining images of lipid droplets (labeled with BODIPY in green as indicator of adipogenic differentiation) in mLPS1 and mLPS1ΔNICD cells after treated with adipocyte differentiation medium, visualized by immunofluorescence microscopy. Scale bars, 50 μm. Nuclei were counterstained with DAPI (blue). C RT-qPCR analysis showing relative levels of mature adipocyte markers in mLPS1 cells at 24 h after expose to adipocyte differentiation medium. D RT-qPCR analysis of mature adipocyte marker expression levels in mLPS1ΔNICD cells at 24 h after expose to adipocyte differentiation medium. Data are represented as mean ± SD. *P < 0.05, **P < 0.001. n.s., not significant.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Quantitative RT-PCR, Expressing, Marker, Staining, Labeling, Immunofluorescence, Microscopy

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A RT-qPCR analysis of mitochondrial DNA (MT-ND1) versus nuclear DNA (HK2) content in mLPS1 and mLPS1ΔNICD cells (n = 3). B mLPS1 and mLPS1ΔNICD cells were stained with the MitoTracker Red FM probe and analyzed by Flow cytometry (n = 3). Histograms show the fluorescence intensity corresponding to mitochondrial mass (left) and quantification of high MitoTracker signal cells (right). C Glucose uptake of mLPS1 and mLPS1ΔNICD cells based on measuring reduction of glucose in culture media over 6 h (n = 4). D lactate production (n = 6) of mLPS1 and mLPS1ΔNICD cells based on measuring lactate concentration in culture media over 16 h. E ATP production (n = 6) of mLPS1 and mLPS1ΔNICD cells based on measuring chemical luminescence signaling in cell after 20 min incubation with reaction buffer. F Cellular respiration was monitored using the Seahorse bioscience extracellular flux analyzer (Left). The oxygen consumption rate (OCR) was normalized to protein abundance. The OCR corresponding to basal respiration (middle) and maximal respiratory capacity (right) in mLPS1 and mLPS1ΔNICD cells were shown (n = 5). G Seahorse bioscience extracellular flux analysis of extracellular acidification rate (ECAR) in mLPS1 and mLPS1ΔNICD cells (left). The basal glycolysis (middle) and maximal glycolytic capacity (right) were quantified in bar graphs (n = 5). Data are presented as mean ± SD of three reading cycles, for each cycle n = 5, *P < 0.05, **P < 0.001.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Quantitative RT-PCR, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Incubation, Quantitative Proteomics

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A Venn diagram showing the overlap of differentially expressed genes in mLPS1 vs mLPS1ΔNICD cells (green) and NICDOE LPS vs adipose tissues (red). Co-upregulated genes represent those suppressed by NICD highly expressed in adipocytes, and the co-downregulated genes represent those activated by NICD highly expressed in LPS cells. B Volcano plot of differentially expressed genes in mLPS1 and mLPS1ΔNICD cells and those co-upregulated or co-downregulated in A. C Heatmap visualization of the co-upregulated and co-downregulated genes. D Heatmap showing mitochondrial metabolic genes regulated by NICD. E Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the top 10 activated pathways in mLPS1ΔNICD cells compared to mLPS1 cells. Results of KEGG and GO analysis were determined to be significant by using a criterion of FDR.Q < 0.05 and a P < 0.05 is shown. F Kaplan–Meier curve of overall survival of sarcoma patients based on PPARGC1A transcripts per million (TPM), data from TCGA database (BMC Cancer, 2014) analyzed with the PROGgeneV2 platform. G Bar chart showing PPARGC1A expression in human liposarcoma tissues (Genevestigator) ranked by expression level from high (Top) to low (Bottom). Different types of human liposarcomas and neoplasms are shown on the left-hand side. Tumor tissue from the same region and source were grouped in the same color marker for comparison. The numbers of samples are listed on the right. Error bars represent standard deviation.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Expressing, Chromosome Transmission Fidelity Colony Color Assay, Comparison, Standard Deviation

Journal: Oncogene

Article Title: Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma

doi: 10.1038/s41388-023-02768-6

Figure Lengend Snippet: A Ppargc1a mRNA levels in mouse inguinal white adipose tissue, brown adipose tissue, TetO-mPGC1αOE stably transfected 293 T cells, and TetO-mPGC1αOE stably transfected mLPS1 cells with or without doxycycline (Dox) induction (n = 3). B PGC-1α protein levels in TetO-mPGC1αOE 293 T and mLPS1 cells treated with or without Dox. C RT-qPCR analysis of mitochondrial DNA (MT-ND1) and nuclear DNA (HK2) in TetO-Empty vector and TetO-mPGC1αOE mLPS1 cells with Dox treatment (n = 3). D TetO-Empty vector and TetO-mPGC1αOE mLPS1 cells were stained with the MitoTracker Red FM probe and analyzed by flow cytometry (n = 3). E The oxygen consumption rate (OCR) was measured with Seahorse in TetO-Empty vector and TetO-mPGC1αOE mLPS1 cells treated with Dox. OCR corresponding to basal mitochondrial respiration (middle) and maximal mitochondrial respiratory capacity (right) were shown (n = 5). F The extracellular acidification rate (ECAR) of TetO-Empty vector and TetO-mPGC1αOE mLPS1 cells treated with Dox. ECAR corresponding to basal glycolysis (middle) and glycolytic capacity (right) were shown (n = 5). Data are presented as mean ± SD of three reading cycles for each cycle, n = 5, *P < 0.05, **P < 0.001.

Article Snippet: Vector construction and cell transfection The doxycycline-inducible TetO-PGC1α OE mLPS1 and empty vector control cells were generated using the Sleeping Beauty transposon system (Addgene #60497).

Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Plasmid Preparation, Staining, Flow Cytometry

Journal: Cell

Article Title: The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity

doi: 10.1016/j.cell.2018.03.070

Figure Lengend Snippet: Diversity and Expression of Type I IFN Genes in R. aegyptiacus , Related to Figure 5 (A) Phylogeny of R. aegyptiacus type I IFN proteins. Maximum likelihood phylogenetic tree of bat type I IFN proteins. Bootstrap evidence (percentage of 500 bootstrap replicates) is labeled on branches if over 65. (B) Antiviral effect of recombinant R. aegyptiacus IFN-ω4. RoNi cells were treated with recombinant IFN-ω4 (rIFN-ω4), rIFN-β1, or an unrelated protein (rPA-D1) for 4 hr, infected with VSV-eGFP at an MOI of 0.05, and imaged for eGFP expression 1 day post infection. Higher concentrations of recombinant IFN-ω4 inhibit viral replication as demonstrated by the absence of eGFP expression in cells after multiple viral replication cycles. Brightness was increased by 20% on all images. (C) Sendai virus (SeV) infection of RoNi cells elicits an IFN response, including IFN-ω. RoNi cell monolayers were infected with SeV strain Cantell at an MOI of 1.0 or mock infected, and harvested for total RNA extraction and sequencing at 3, 8, and 24 hr. Sequencing data were quantified by IFN subtype in transcripts per million (TPM). Values plotted are the mean ± standard deviation of three replicates for each time point. IFN-ε and IFN-δ were not expressed. Adj. p values from unpaired t test between SeV and mock: ∗ < 0.05, ∗∗ < 0.005, ∗∗∗ < 0.0005. See .

Article Snippet: TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat High Throughput (96 samples, 96 indexes) , Illumina , Cat# 20020597.

Techniques: Expressing, Labeling, Recombinant, Infection, Virus, RNA Extraction, Sequencing, Standard Deviation

Journal: Cell

Article Title: The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity

doi: 10.1016/j.cell.2018.03.070

Figure Lengend Snippet:

Article Snippet: TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat High Throughput (96 samples, 96 indexes) , Illumina , Cat# 20020597.

Techniques: Virus, Recombinant, Lysis, Purification, DNA Extraction, Library Amplification, Library Quantification, Isolation, Binding Assay, DNA Sequencing, High Throughput Screening Assay, Sequencing, Software